Two secretory luciferases

Abstract

The present invention is one gene construct or a combination of two gene constructs or expression vectors incorporating a Cypridina luciferase gene and a copepod luciferase under the control of distinct promoters. These gene constructs and expression vectors are useful for making a mammalian cell incorporating the Cypridina luciferase gene and the copepod luciferase to be capable of stably expressed and extracellularly secreted under the control of the distinct promoters.

1 . One gene construct or a combination of two gene constructs incorporating a  Cypridina  luciferase gene and a copepod luciferase gene under the control of distinct promoters. 
     
     
         2 . One expression vector or a combination of two expression vectors incorporating a  Cypridina  luciferase gene and a copepod luciferase gene under the control of distinct promoters. 
     
     
         3 . A mammalian cell incorporating a  Cypridina  luciferase gene and a copepod luciferase gene to be capable of being stably expressed and extracellularly secreted under the control of distinct promoters. 
     
     
         4 . The mammalian cell according to  claim 1  wherein one luciferase is under the control of the promoter subjected to evaluation and the other luciferase is under the control of the promoter subjected to comparison. 
     
     
         5 . A method for producing a mammalian cell incorporating a  Cypridina  luciferase gene and a copepod luciferase gene to be capable of being stably expressed and extracellularly secreted under the control of distinct promoters, characterized in that the gene construct or the combination thereof according to  claim 1  or the expression vector or the combination thereof according to  claim 2  is introduced into the mammalian cell. 
     
     
         6 . A kit for detecting dual gene expression of a  Cypridina  luciferase gene and a copepod luciferase gene incorporated into a mammalian cell, comprising  Cypridina  luciferin and coelenterazine. 
     
     
         7 . The kit according to  claim 6  comprising a  Cypridina  luciferin solution and a coelenterazine solution. 
     
     
         8 . The kit according to  claim 6  comprising a  Cypridina  luciferin solution containing  Cypridina  luciferin and at least one antioxidant and a coelenterazine solution containing coelenterazine and at least one antioxidant, wherein the antioxidant combined with the  Cypridina  luciferin is selected from the group consisting of ascorbic acid or salts thereof, erythorbic acid or salts thereof, or sulfite salts and the antioxidant combined with coelenterazine is selected from the group consisting of ascorbic acid or salts thereof, erythorbic acid or salts thereof, or sulfite salts. 
     
     
         9 . The kit according to  claim 6  having a solution containing  Cypridina  luciferin and an ascorbate salt and a solution containing coelenterazine and an ascorbate salt. 
     
     
         10 . The kit according to  claim 6  having a solution containing  Cypridina  luciferin, an ascorbate salt and a sulfite salt and a solution containing coelenterazine, an ascorbate salt and a sulfite salt. 
     
     
         11 . The kit according to  claim 6  having a solution containing  Cypridina  luciferin, sodium ascorbate and sodium sulfite and a solution containing coelenterazine, sodium ascorbate and sodium sulfite. 
     
     
         12 . A method for screening drugs comprising a step of culturing the mammalian cell according to  claim 3  in the presence of a drug candidate compound, a step of quantifying  Cypridina  luciferase and copepod luciferase extracellularly secreted in the presence or absence the candidate compound in the presence of  Cypridina  luciferin or coelenterazine, respectively, and a step of evaluating an effect of the candidate compound on a promoter subjected to evaluation ligated to at least one luciferase gene. 
     
     
         13 . A system of measuring multiple transcription activities of respective promoters ligated to respective luciferases before and after the change of a culture environment by changing the culture environment of the mammalian cell according to [3] and evaluating expression amounts of two luciferases.